CN107743400A - For treating the therapeutic combination and method of neoplasia - Google Patents

Abstract

It is the method for treating solid tumor provided herein, these methods include giving MEDI4736 or its antigen-binding fragment, the Sibutramine Hydrochloride wood monoclonal antibody or its antigen-binding fragment and MEDI6383 of effective dose.

Description

For treating the therapeutic combination and method of neoplasia

Sequence table

The application is included the sequence table electronically submitted with ASCII fromat and tied hereby with its full text by quoting Close.The ASCII copy creatings were 14 days, entitled BTO-200WO1_SL.txt April in 2016, and size is 34,448 Byte.

Background technology

Cancer continues to be main Global Health burden.Although being in progress in terms of the treatment of cancer, exist always To the unsatisfied needs of medical treatment of more effective and smaller toxicity therapy, particularly with the evening resistant to existing therapeutic agent Those patients of phase disease or cancer.

The cytotoxicity of immune system, particularly T cell mediation, the effect in tumour control is generally acknowledged.Have more next More evidences shows that T cell controls tumour growth and survival in cancer patient, either in the early and late of the disease Stage.However, tumour-specific T- cell responses are difficult to rise and maintain in cancer patient.

The T cell path significantly paid close attention to so far by toxic T lymphocyte antigen -4 (CTLA-4, CD152), Programmed death ligand 1 (PD-L1, also referred to as B7-H1 or CD274) and OX40 (CD134；TNFRSF4) signal.

CTLA4 is expressed in the T cell of activation, and with CD28 mediations t cell activation as co-suppression agent to suppress T Cell response.CTLA4 is considered as activating amplitude early stage adjusting inmature and memory t cell after TCR engagements, and is shadow Ring a part for the central suppression approach of both antineoplastic immune and autoimmunity.CTLA4 is specially expressed in T cell, and Its part CD80 (B7.1) and CD86 (B7.2) expression is limited primarily to antigen presenting cell, T cell and other are immune-mediated Cell.It has been reported that block the Antagonism anti-CTLA 4 antibody enhancing t cell activation of CTLA4 signal paths.Resist as one The easy Puli's nurse agate of body was ratified by FDA the treatment for metastasis melanin tumor in 2011.Another anti-CTLA 4 antibody Sibutramine Hydrochloride wood Monoclonal antibody, the treatment for advanced melanoma is tested in III clinical trial phases, but compared to standard care at that time (for not Azoles amine or Dacarbazine) do not dramatically increase the overall survival of patient.

PD-L1 is also a part for the complication system of the acceptor and part that are related to control T- cell-stimulatings.In normal structure In, PD-L1 is in T cell, B cell, BMDC, macrophage, mescenchymal stem cell, bone marrow derived mast cell and not With being expressed on non-hematopoietic cell.Its normal function be by two recipient programs dead 1 with it (also known as PD-1 or CD279) and CD80 (also known as B7-1 or B7.1) interaction adjusts the balance between T- cell-stimulatings and tolerance.PD- L1 is also expressed and worked on multiple positions to help tumour to avoid by the inspection of host immune system progress by tumour Survey and eliminate.PD-L1 wide scope cancer high frequency express.In certain cancers, PD-L1 expression and survival reduce It is associated with unfavorable prognosis.Block the interaction between B7-H1 and its acceptor antibody can release in vitro PD-L1 according to Rely property immunosuppressive action and strengthen the cellular cytoxicity activity of antitumor T cell.MEDI4736 be for human PD-L 1 can Block the human monoclonal antibodies of the combination of both PD-L1 and PD-1 and CD80 acceptors.

OX40 is to be mainly seen in activation CD4+ and CD8+T cells, regulatory T-cell (Treg) and NKT (NK) cell On a kind of Tumor Necrosis Factor Receptors (TNFR).Being signaled on CD4+ the and CD8+T cells of activation by OX40 causes carefully Enhancing, granzyme and perforin release and the amplification in effect and memory T cell pond caused by intracellular cytokine.It is in addition, thin in Treg OX40 signal transductions on born of the same parents suppress Treg amplification, close the induction to Treg and block Treg to suppress function.

Significant progress was made that in developing for the strategy of anticancer and other diseases although in the past 10 years, but It is that there is limited selection of clinical with late period, intractable and metastatic disease patient.Chemotherapy, radiation and high dose chemotherapy Turn into dosage to limit.There are still to the less method of new toxicity and with it is more preferable the effect of, longer clinical benefit and A large amount of unsatisfied demands of the treatment of improved safety features, especially for resistant to existing treatment Those of terminal illness or cancer patient.

Summary of the invention

On the one hand, the method for treating solid tumor in subject (for example, human experimenter) the invention provides one kind, This method includes giving anti-PD-L1 antibody (for example, MEDI4736) or its antigen-binding fragment, anti-CTLA-4 to the subject Antibody (for example, Sibutramine Hydrochloride wood monoclonal antibody) or its antigen-binding fragment and OX40 activators (for example, MEDI6383).

On the other hand, the invention provides the side of one kind treatment solid tumor in subject (for example, human experimenter) Method, this method include giving MEDI4736 or its antigen-binding fragment, Sibutramine Hydrochloride wood monoclonal antibody or its antigen binding fragment to the subject Section and MEDI6383.

On the other hand, the invention provides a kind of pharmaceutical composition, the pharmaceutical composition contains the anti-PD- of effective dose L1 antibody (for example, MEDI4736) or its antigen-binding fragment, anti-CTLA-4 antibody (for example, Sibutramine Hydrochloride wood monoclonal antibody) or its antigen Binding fragment and OX40 activators (for example, MEDI6383) and pharmaceutically acceptable excipient.

On the other hand, the invention provides a kind of pharmaceutical composition, the pharmaceutical composition to contain effective dose MEDI4736 or its antigen-binding fragment, Sibutramine Hydrochloride wood monoclonal antibody or its antigen-binding fragment and MEDI6383 or its active fragment, with And pharmaceutically acceptable excipient.

On the other hand, the invention provides a kind of kit, the kit containing it is with good grounds it is as described herein any other The pharmaceutical composition of aspect and for treating cancer specification (for example, according to the side as described herein in terms of any other Method).

In the various embodiments of any aspect as described herein, the OX40 activators are that one or more OX40 parts melt Hop protein (for example, MEDI6383) or anti-OX40 antibody.

In the various embodiments of any aspect as described herein, the anti-PD-L1 antibody is MEDI4736.

In the various embodiments of any aspect as described herein, the anti-CTLA-4 antibody is Sibutramine Hydrochloride wood monoclonal antibody.

In the various embodiments of any aspect as described herein, these, which are given, increases survival period.In various embodiments In, with independent MEDI4736, independent Sibutramine Hydrochloride wood monoclonal antibody or individually compared with the giving of MED6383, these, which are given, causes survival period Increase.In a further embodiment, it is single with MEDI4736 and Sibutramine Hydrochloride wood monoclonal antibody, MEDI4736 and MEDI6383 and Sibutramine Hydrochloride wood Anti- to be compared with giving for MEDI6383, these give the increase for causing survival period.

In the various embodiments of any aspect as described herein, these, which are given, reduces gross tumor volume.In various implementations In example, with independent MEDI4736, independent Sibutramine Hydrochloride wood monoclonal antibody or individually compared with the giving of MED6383, these, which are given, causes tumour body Long-pending reduction.In a further embodiment, with MEDI4736 and Sibutramine Hydrochloride wood monoclonal antibody, MEDI4736 and MEDI6383 and Sibutramine Hydrochloride Wooden monoclonal antibody is compared with giving for MEDI6383, and these give the reduction for causing gross tumor volume.

In the various embodiments of any aspect as described herein, anti-PD-L1 antibody (for example, MEDI4736) or it is anti- Giving for former binding fragment is carried out by intravenous infusion.It is anti-in the various embodiments of any aspect as described herein CTLA-4 antibody (for example, Sibutramine Hydrochloride wood monoclonal antibody) or giving for its antigen-binding fragment are carried out by intravenous infusion.At this In the various embodiments of any aspect described in text, OX40 activators (for example, MEDI6383) or giving for its active fragment are Carried out by intravenous infusion.In the various embodiments of any aspect as described herein, the pharmaceutical composition is formulated For intravenous administration.

In the various embodiments of any aspect as described herein, the solid tumor is oophoroma, breast cancer (for example, three is cloudy Property breast cancer), colorectal cancer, prostate cancer, cervical carcinoma, uterine cancer, carcinoma of testis, carcinoma of urinary bladder, head and neck cancer, melanoma, pancreas One kind or more in gland cancer, clear-cell carcinoma, lung cancer or non-small cell lung cancer (for example, squamous or non-squamous non-small cell lung cancer) Kind.In the different embodiments of any aspect described here, the subject is people patient.

Definition

Unless otherwise defined, all technologies used herein and scientific terminology have those skilled in the art in the invention The implication being generally understood that.Following bibliography provides the generic definition of multiple terms used in the present invention for technical staff： Singleton et al., Dictionary of Microbiology and Molecular Biology [microbiology and molecule Biology dictionary] (second edition, 1994)；The Cambridge Dictionary of Science and Technology [Cambridge science and technology dictionary) (Walker is edited, 1988)；The Glossary of Genetics [science of heredity vocabulary], 5th edition, Rieger et al. (editor), Springer Verlag [Springer Verlag] (1991)；And Hale with Marham, The Harper Collins Dictionary of Biology [Harper Collins's biology dictionary] (1991 Year).Except as otherwise noted, following term as used herein has following their implication of imparting.

" antitumor activity " means the propagation of any reduction or stable tumour cell or the biological activity of survival.At one In embodiment, the antitumor activity is anti-tumor immune response.

" immunomodulator " means to strengthen the reagent of immune response (such as anti-tumor immune response).The present invention's is exemplary Immunomodulator includes antibody, such as anti-CTLA-4 antibody, anti-PD-L1 antibody and its fragment；And protein, such as OX40 parts Fusion protein or its fragment.In one embodiment, the immunomodulator is immunologic test point inhibitor.

" OX40 polypeptides " mean with NCBI accession number NP_003318 have at least about the polypeptide of 85% amino acid identity or Its fragment.OX40 is on the surface of Antigen-activated mammal CD4+ and CD8+T lymphocyte in acceptor TNFR superfamilies The member of upper expression.See, e.g., Paterson et al., Mol Immunol [molecular immunology] 24,1281-1290 (1987)；Mallett et al., EMBOJ [European Molecular Bioglogy Organization's magazine] 9,1063-1068 (1990)；With Calderhead et al., JImmunol [Journal of Immunology] 151,5261-5271 (1993).OX40 is also known as CD134, ACT- 4 and ACT35.OX40 receptor sequences are as known in the art and for example with Genbank accession numbers：AAB33944 or CAE11757 is provided.

The following provide exemplary people OX40 amino acid sequences (SEQ ID NO：18)：

" OX40 parts " means there is at least about 85% amino acid identity and special with NCBI accession number NP_003317 The polypeptide or its fragment of property combination OX40 acceptors.See, e.g., Baum P.R. et al. EMBO J [European Molecular Bioglogy Organizations Magazine] .13：3992-4001(1994)).Term OX40L include whole OX40 parts, soluble OX40 parts and comprising The fusion protein of the functional activity part for being covalently linked to Part II such as protein structure domain of OX40 parts.It is additionally included in In OX40L definition is that different still reservations are specifically bound to OX40 on amino acid sequence from naturally occurring OX40L The variant of the ability of acceptor.Be additionally included in OX40L definition be strengthen OX40 bioactivity variant.OX40 part sequences Row are well known in the art and for example with Genbank accession number：NP_003318 is provided.

The following provide exemplary people OX40 ligand amino acid sequences (SEQ ID NO：19)：

MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEK GFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVY LNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL

" OX40 activators " means that the bioactivity of OX40 acceptors is interacted and increased with OX40 receptor-specifics OX40 parts.It is desirable to, the bioactivity increase at least about 10%, 20%, 30%, 50%, 70%, 80%, 90%th, 95% or even 100%.In some aspects, OX40 activators as disclosed in this include OX40 Binding peptides, such as The fragment or derivative of anti-OX40 antibody (for example, OX40 agonist antibodies), OX40 parts or these molecules.

" OX40 antibody " means to specifically bind OX40 antibody.OX40 antibody includes having specific Dan Ke to OX40 Grand antibody and polyclonal antibody and its antigen-binding fragment.In some aspects, anti-OX40 antibody as described herein is Dan Ke Grand antibody (or its antigen-binding fragment), such as muroid, humanization or human monoclonal antibodies.In a specific embodiment, The OX40 antibody is OX40 receptor stimulating agents, such as by Weinberg (Weinberg) et al., immunological therapy magazine (J Immunother) the mouse anti human OX40 monoclonal antibodies (9B12) described by 29,575-585 (2006).In other embodiment In, it is specifically bound to OX40 antibody or its antigen-binding fragment is attached to and mAb 9B12 identical OX40 epitopes.

" OX40 ligand fusion proteins (OX40L FP) " means to specifically bind OX40 acceptors and increases the egg of immune response White matter.In one embodiment, OX40 ligand fusion proteins are attached to OX40 acceptors by strengthening T cell identification to strengthen tumour Antigen-specific immune response.Exemplary OX40 ligand fusion proteins are in entitled " tripolymer OX40 immunoglobulins fusion egg White and application method " (" Trimeric OX40 Immunoglobulin Fusion Protein and Methods of Use ") United States Patent (USP) 7,959,925 in be described.See, e.g., the ID NO.8 of United States Patent (USP) 7,959,925, SEQ (SEQ ID NO：20)：

LATDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKELLGGGSIKQIEDKIEEILSKIYHIENEIARIKKLIGERGHGGGSNSQVSHRYPRFQSIKVQFTEYKKEKGFIL TSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVT Other OX40 ligand fusion proteins of TDNTSLDDFHVNGGELILIHQNPGEFCVL are for example in U.S. Patent number 6,312,700 It is described.In one embodiment, OX40 ligand fusion proteins enhancing tumor specific T cells immunity.In an implementation In example, OX40 ligand fusion proteins are MEDI6383.

" PD-L1 polypeptides " mean with NCBI accession number NP_001254635 have at least about 85% amino acid identity and Polypeptide or its fragment with PD-1 and CD80 binding activity.

" PD-L1 nucleic acid molecules " mean to encode the polynucleotides of PD-L1 polypeptides.Exemplary PD-L1 sequence of nucleic acid molecules with NCBI accession number NM_001267706 is provided.

" anti-PD-L1 antibody " means the antibody of selective binding PD-L1 polypeptides.Exemplary anti-PD-L1 antibody is described in Such as US 20130034559/US 8779108 and US 20140356353, it is incorporated herein by reference.MEDI4736 is to show The example anti-PD-L1 antibody of property.Other anti-PD-L1 antibody include BMS-936559 (Mei Shiguibao companies (Bristol-Myers )) and MPDL3280A (Roche Holding Ag (Roche)) Squibb.

MEDI4736 VL(SEQ ID NO：1)

EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFT LTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK

MEDI4736 VH(SEQ ID NO：2)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS

MEDI4736 VH CDR1(SEQ ID NO：3)

RYWMS

MEDI4736 VH CDR2(SEQ ID NO：4)

NIKQDGSEKYYVDSVKG

MEDI4736 VH CDR3(SEQ ID NO：5)

EGGWFGELAFDY

MEDI4736 VL CDR1(SEQ ID NO：6)

RASQRVSSSYLA

MEDI4736 VL CDR2(SEQ ID NO：7)

DASSRAT

MEDI4736 VL CDR3(SEQ ID NO：8)

QQYGSLPWT

" CTLA-4 polypeptides " means the consensus amino acid sequence for having at least 85% with Genbank accession number AAL07473.1 The polypeptide of property or its there is the fragment of T cell inhibitory activity.The following provide AAL07473.1 sequence (SEQ ID NO：21)：

Gi | 15778586 | gb | AAL07473.1 | AF414120_1CTLA-4 [homo sapiens]

MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTV LRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

" CTLA-4 nucleic acid molecules " mean to encode the polynucleotides of CTLA-4 polypeptides.Exemplary CTLA-4 polynucleotides are with base Because storehouse accession number AAL07473 is provided.

" anti-CTLA-4 antibody " means the antibody of selective binding CTLA-4 polypeptides.Exemplary anti-CTLA-4 antibody is retouched It is set forth in such as U.S. Patent number 6,682,736；7,109,003；7,123,281；7,411,057；7,824,679；8,143, 379；7,807,797；With 8, in 491,895 (wherein, Sibutramine Hydrochloride wood monoclonal antibody is 11.2.1), these patents are hereby incorporated by reference This.Sibutramine Hydrochloride wood monoclonal antibody is exemplary anti-CTLA-4 antibody.The following provide the sequence of Sibutramine Hydrochloride wood monoclonal antibody.

Sibutramine Hydrochloride wood monoclonal antibody, U.S. Patent number 6,682,736

Sibutramine Hydrochloride wood monoclonal antibody VL (SEQ ID NO：9)

PSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

Sibutramine Hydrochloride wood monoclonal antibody VH (SEQ ID NO：10)

GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVH

Sibutramine Hydrochloride wood monoclonal antibody VH CDR1 (SEQ ID NO：11)

GFTFSSYGMH

Sibutramine Hydrochloride wood monoclonal antibody VH CDR2 (SEQ ID NO：12)

VIWYDGSNKYYADSV

Sibutramine Hydrochloride wood monoclonal antibody VH CDR3 (SEQ ID NO：13)

DPRGATLYYYYYGMDV

Sibutramine Hydrochloride wood monoclonal antibody VL CDR1 (SEQ ID NO：14)

RASQSINSYLD

Sibutramine Hydrochloride wood monoclonal antibody VL CDR2 (SEQ ID NO：15)

AASSLQS

Sibutramine Hydrochloride wood monoclonal antibody VL CDR3 (SEQ ID NO：16)

QQYYSTPFT

The term " antibody " such as used in present disclosure refers to immunoglobulin or its fragment or derivative, and covers bag Any polypeptide of antigen binding site is included, no matter it is produced in vitro or in vivo.The term includes but is not limited to：More grams Grand, monoclonal, monospecific, polyspecific, non-specificity, humanization, single-stranded, chimeric, synthesis, restructuring, heterozygosis, mutation, with And bound antibody.Unless modified in addition with term " complete ", it is " anti-for the purpose of present disclosure, term such as in " complete antibody " Body " also includes antibody fragment such as Fab, F (ab ')₂, Fv, scFv, Fd, dAb and retain antigen binding function (specifically bind Such as CTLA-4 or PD-L1 ability) other antibody fragments.Typically, this kind of fragment will include antigen-binding domains.

Term " antigen-binding domains ", " antigen-binding fragment " and " binding fragment " refers to include being responsible for antibody and antigen Between the part of the antibody molecule of amino acid that specifically binds.In the case where antigen is very big, antigen-binding domains can To be only joined to a part for the antigen.It is responsible for the part with the antigen molecule of antigen-binding domains specificity interaction It is referred to as " epitope " or " antigenic determinant ".Antigen-binding domains typically comprise antibody light chain variable region (V_L) and antibody weight Chain variable region (V_H), however, it must not necessarily include both.For example, so-called Fd antibody fragments are only by V_HDomain forms, But still retain some antigen binding functions of complete antibody.

The binding fragment of antibody produces by recombinant DNA technology or by the enzymatic of complete antibody or chemical cracking.Knot Closing fragment includes Fab, Fab ', F (ab ') 2, Fv and single-chain antibody.In addition to " bispecific " or " difunctional " antibody, resist It is identical that body, which is interpreted as each of which binding site,.Using enzyme (papain) come to digest the result of antibody be two identical Antigen-binding fragment, also known as " Fab " fragment and " Fc " fragment, they are without antigen-binding activity but with the energy of crystallization Power.With enzyme (pepsin) come to digest the result of antibody be the fragments of F (ab ') 2, wherein the two of the antibody molecule arm keeps link And including two antigen binding sites.The fragments of F (ab ') 2 have the ability of crosslinking antigen.When used herein, " Fv " refers to Remain the minimal segment of the antibody of antigen recognizing and antigen binding site.When used herein, " Fab " refers to include light chain The fragment of the antibody of the CHI domains of constant domain and heavy chain.

Term " mAb " refers to monoclonal antibody.The antibody of the present invention includes but is not limited to：All Pure Nature antibody, bispecific Antibody；Chimeric antibody；Fab, Fab ', Single chain V region fragments (scFv), fused polypeptide and unconventional antibody.

In this disclosure, " including (comprises, comprising) ", " including (containing) ", " have " etc. (having) there is United States patent law to assign their implication and can mean " including (includes, including) " Deng；" substantially by ... form (consisting essentially of or consists essentially) " equally has The implication and the term that United States patent law assigns are open, it is allowed to beyond the presence described, as long as the base described This or novel feature are not exceeded the presence change of narration, but exclude prior embodiment.

As used herein term " it is determined that ", " evaluation ", " measure ", " measurement " and " detection " refer to it is qualitatively and quantitatively true It is fixed, and just because of this, term " it is determined that " be used interchangeably herein with " measure ", " measurement " etc..For the purpose of wherein quantitatively determining When, use " determination of amount " of phrase analysis thing and the like.It is wherein qualitative and/or quantitatively determine for purpose when, use is short Language " level for determining analyte " or " detection " analyte.

" reference " means the standard compared.

" subject " means mammal, including but not limited to people or non-human mammal, such as ox, horse, dog, sheep or cat.

Writing a Chinese character in simplified form to all values in the range of this is understood as that in the scope that this is provided.For example, the scope of one 1 to 50 It should be understood to include any numeral from the following group, number combinatorics on words or subrange, the group to be made up of the following：1、2、3、 4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、 31st, 32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50.

Term " treatment (treat, treating, treatment etc.) " as used in this refers to reduce or improves barrier Hinder and/or relative symptom.It will be understood that although can not exclude, treat obstacle or illness has been not required for The obstacle, illness or relative symptom are eliminated entirely.

Unless explicitly claimed or from context it is clear that as used in this, term "or" is understood to include It is interior.Unless explicitly claimed or from context it is clear that as used in this, term " a kind of (a) ", " one (an) " and " being somebody's turn to do (the) " is understood to odd number or plural.

Unless definite provide or from context it is clear that otherwise term " about (about) " as used in this is managed Solve as in the normal tolerance range of this area, for example, within 2 standard deviations of average.About it is construed as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or the 0.01% of statement value Within.Unless from context it is clear that all numerical value are about modified by the term provided herein.

It is any single that narration in any definition of this variable to chemical group inventory, which is included the variable-definition, The combination of group or listed group.To this variable or the embodiment of aspect narration include as any single embodiment or with The embodiment that any other embodiment or part thereof combines.

Can be by any combinations thing provided herein or method and provided herein in any other composition and method One or more are combined.

Brief Description Of Drawings

Figure 1A -1F are shown, compared to the monotherapy and double combinations therapy of medicament, with anti-CTLA-4 antibody, anti-PD-L1 The combined therapy of antibody and OX40 ligand fusion proteins is more effective in terms of gross tumor volume and increase survival period is reduced.Figure 1A is Describe figure of the seminar with the mean tumour volume of time.Figure 1B display describe isotype controls (left figure) and untreated by Examination person's (right figure) with the single gross tumor volume of time figure.Fig. 1 C displays are described with anti-CTLA-4 monoclonal antibodies (CTLA- 4mAb；Left figure)；Anti- PD-L1 monoclonal antibodies (PD-L1 mAb；Middle figure)；With OX40 ligand fusion proteins (mOX40L FP；It is right Figure) treatment subject with the single gross tumor volume of time figure.Fig. 1 D displays are described with anti-CTLA-4 and anti-PD-L1 Dan Ke Grand antibody (CTLA-4 mAb+PD-L1mAb；Top, left figure)；Anti- PD-L1 monoclonal antibodies and OX40 (PD-L1 mAb+ mOX40L FP；Top, right figure)；With anti-CTLA-4 monoclonal antibodies and OX40 ligand fusion proteins (CTLA-4 mAb+ mOX40L FP；Bottom, left figure)；And anti-CTLA-4 and anti-PD-L1 monoclonal antibodies and OX40 (CTLA-4 mAb+PD-L1 MAb+mOX40L FP) combined therapy subject with the single gross tumor volume of time figure.Fig. 1 E are shown in untreated , isotype controls, CTLA-4 mAb, PD-L1 mAb and mOX40L FP seminar with the percentage survival of time figure.Figure 1F is shown in CTLA-4 mAb+PD-L1 mAb, isotype controls, CTLA-4mAb+mOX40L FP, PD-L1 mAb+ With the percentage survival of time in mOX40L FP and CTLA-4 mAb+PD-L1 mAb+mOX40L FP FP seminar Figure.11 C57BL/6 mouse were in the 1st day subcutaneous vaccination (SC) MCA205 cell in each group.In the 11st, 15,18 and 22 day abdomen (IP) control sample (isotype controls) and test sample anti-CTLA-4 mAb and anti-PD-L1 mAb are given in film；The 11st Test sample mOX40L FP are given with 15 days intraperitoneals.To test sample treatment between the animal of isotype controls treatment Compare, and chart board prism (GraphPad Prism) 6.0 softwares are used by the method described in the 6.6th chapters and sections Analyze the statistical significance of group difference.Error bars represent the standard error of average value.IP=intraperitoneals；SC=is subcutaneous 's；TGI=Tumor growth inhibitions.

Detailed description of the invention

The invention is characterized in that the composition useful to treating cancer and method, it includes anti-CTLA-4 antibody, anti-PD- The combination of L1 antibody and OX40 activators (for example, OX40 ligand fusion proteins).As reported herein below, with these medicines The treatment of agent reduces gross tumor volume in mouse tumor model and adds survival period.

It there is provided herein the method for treating solid tumor.The method provided include give effective dose MEDI4736 or Its antigen-binding fragment, Sibutramine Hydrochloride wood monoclonal antibody or its antigen-binding fragment and OX40 ligand fusion proteins or its active fragment.Each The solid tumor is in kind embodiment, but is not limited to oophoroma, breast cancer (for example, triple negative breast cancer), colorectal cancer, forefront Gland cancer, cervical carcinoma, uterine cancer, carcinoma of testis, carcinoma of urinary bladder, head and neck cancer, melanoma, cancer of pancreas, clear-cell carcinoma and lung cancer (example Such as, non-small cell lung cancer (NSCLC)).There are three main NSCLC hypotypes：Squamous cell carcinoma, gland cancer and maxicell (undifferentiated) Cancer.Other hypotypes include adenosquamous carcinoma and carcinoma sarcomatodes.

Anti-tumor therapy

The method for treating cancer is there is provided herein, these methods are including giving anti-CTLA 4 antibody, anti-PD-L1 resists Body and OX40 activators (for example, OX40 ligand fusion proteins, OX40 agonist antibodies).Give anti-CTLA 4 antibody, anti-PD- L1 antibody and OX40 ligand fusion proteins cause the reduction of gross tumor volume and the increase of survival period in mouse tumor model. Some aspects, anti-CTLA 4 antibody (for example, Sibutramine Hydrochloride wood monoclonal antibody), anti-PD-L1 are given to the patient with solid tumor And OX40 ligand fusion proteins (for example, MEDI6383) (MEDI4736).In some aspects, according to these sides provided herein Method, anti-CTLA 4 antibody (for example, Sibutramine Hydrochloride wood monoclonal antibody), anti-PD-L1 (MEDI4736) and OX40 ligand fusion proteins (for example, Giving MEDI6383) is by parenteral (for example, intravenous infusion or hypodermic injection).In some aspects, by anti-CTLA 4 Antibody (for example, Sibutramine Hydrochloride wood monoclonal antibody), anti-PD-L1 (MEDI4736) and OX40 ligand fusion proteins (for example, MEDI6383) are made Given for single pharmaceutical composition.

Use effective treatment bag of cancer therapy (including anti-CTLA 4 antibody, anti-PD-L1 antibody and OX40 activators) Include, for example, at primary tumor site or in one or more transfers, reduce progression rates, the retardance or stable swollen of cancer Knurl or transforming growth, actual shrinkage, and/or tumor regression.In certain aspects, tumour growth reduction or retardance can be system Meter is learned upper significant.Can by the growth with the patient tumors at baseline, for expected tumour growth, for based on big The expection tumour growth of PATIENT POPULATION or tumour growth for control population are compared to measure the reduction of tumour growth. In other embodiment, these methods of the invention increase survival.

Face giving cancer therapy (including anti-CTLA 4 antibody, anti-PD-L1 antibody and OX40 ligand fusion proteins) Bed response can be assessed using diagnostic techniques known to clinician, and these diagnostic techniques include but is not limited to magnetic resonance imaging (MRI) cell point of scanning, x- radiographics, computed tomography (CT) scanning, flow cytometry or fluorescence-activation Device (FACS) analysis, histology, Gross pathology and blood chemistry are selected, these clinical responses include but is not limited to pass through The change that ELISA, RIA and chromatography detect.

T cell regulatory pathway

There is increasing evidence that T cell controls tumour growth and survival in cancer patient, either in the disease The early and late stage of disease.However, tumour-specific T- cell responses are difficult to rise and maintain in cancer patient.

The T cell regulatory pathway significantly paid close attention to passes through toxic T lymphocyte antigen -4 (CTLA-4, CD152), program Property death ligand 1 (PD-L1, also referred to as B7H-1 or CD274) and OX40 (CD134；TNFRSF4) signal.

CTLA-4 is expressed in the T cell of activation, and with CD28 mediations t cell activation as co-suppression agent to suppress T cell response.CTLA-4 is considered as activating amplitude early stage adjusting inmature and memory t cell after TCR engagements, and is Influence a part for the central suppression approach of both antineoplastic immune and autoimmunity.CTLA-4 is expressed in T cell, and its Part CD80 (B7.1) and CD86 (B7.2) expression is limited primarily to antigen presenting cell, T cell and other are immune-mediated thin Born of the same parents.It is reported that block the anti-CTLA-4 antibody enhancing t cell activation of Antagonism of CTLA-4 signal paths.One such antibody is easy Puli's nurse agate was ratified by FDA the treatment for metastasis melanin tumor in 2011.Another anti-CTLA-4 antibody Sibutramine Hydrochloride wood is single It is anti-, the treatment for advanced melanoma is tested in test in the III phases, but compare at that time standard care (Temozolomide or Dacarbazine) do not dramatically increase the overall survival of patient.

PD-L1 is also a part for the complication system of the acceptor and part that are related to control T- cell-stimulatings.In normal structure In, PD-L1 is in T cell, B cell, BMDC, macrophage, mescenchymal stem cell, bone marrow derived mast cell and not With being expressed on non-hematopoietic cell.Its normal function be by two recipient programs dead 1 with it (also known as PD-1 or CD279) and CD80 (also known as B7-1 or B7.1) interaction adjusts the balance between T- cell-stimulatings and tolerance.PD- L1 is also expressed and worked on multiple positions to help tumour to avoid by the inspection of host immune system progress by tumour Survey and eliminate.PD-L1 wide scope cancer high frequency express.In certain cancers, PD-L1 expression and survival reduce It is associated with unfavorable prognosis.Block the antibody of the interaction between PD-L1 and its acceptor (such as PD-1) can be in vitro Release PD-L1 dependent immunities inhibitory action and strengthen the cellular cytoxicity activity of antitumor T cell.

OX40(CD134；TNFRSF4) be mainly seen in CD4+ the and CD8+T cells of activation, regulatory T-cell (Treg), with A kind of and Tumor Necrosis Factor Receptors (TNFR) (Croft et al., 2009) on NKT (NK) cell.OX40 has one kind Known endogenic ligand OX40 parts (OX40L；CD152；TNFSF4), it exists and can assembled with trimeric form OX40, so as to produce the effective cell signal transduction event (Croft et al., 2009) in T cell.In the CD4+ and CD8+ of activation Being signaled in T cell by OX40 causes enhancing caused by cell factor, granzyme and perforin release and effect and memory The amplification (Jensen et al., 2010) in T cell pond.In addition, OX40 signal transductions on Treg cells suppress Treg amplification, Close induction to Treg and block Treg suppress function (Voo et al., 2013；Vu et al., 2007).

Immunohistochemistry research and early stage flow cytometry show that OX40 is infiltrating the T of extensive human cancer Expressed on cell (Baruah et al., 2011；Curti et al., 2013；Ladanyi et al., 2004；Petty et al., 2002； Ramstad et al., 2000；Sarff et al., 2008；Vetto et al., 1997).OX40 on tumor infiltrating lymphocyte Expression is related to the longer survival period in several human cancer, shows that OX40 signals can be in anti-tumor immune response be established Play an important role (Ladanyi et al., 2004；Petty et al., 2002).

In various non-clinical mouse tumor models, OX40 activator (including antibody and OX40 ligand fusion proteins) is Success uses, have satisfied result (Kjaergaard et al., 2000；Ndhlovu et al., 2001；Weinberg et al., 2000).By OX40 activator costimulations T cell promote antitumor activity be in some cases it is lasting, so as to for Tumour afterwards, which excites, provides lasting protection (Weinberg et al., 2000).The common thorn of Treg- Carbazole alkaloids and effector T cell Swash and be proved to be necessary (Piconese et al., 2008) for the Tumor growth inhibition of OX40 activators.

Anti- PD-L1 antibody

MEDI4736 is exemplary anti-PD-L1 antibody, the antibody it is selective for PD-L1 and blocked PD-L1 with The combination of PD-1 and CD80 acceptors.MEDI4736 can release the suppression to people's T- cell-stimulatings of PD-L1 mediations in vitro, and And suppress tumour growth in xenograft models by T- cell-dependent mechanisms.

On U.S. Patent number can be seen for the information of the MEDI4736 (or its fragment) in method provided herein 8,779,108, its disclosure content is combined herein by quoting with its full text.MEDI4736 crystallizable (Fc) domain of fragment exists Triple mutant is included in the constant domain of IgG1 heavy chains, the triple mutant is reduced with being responsible for the mediation of mediate antibody dependent cell Cytotoxicity (ADCC) complement component C1q and Fc γ acceptors combination.

For the MEDI4736 in these methods provided herein and its antigen-binding fragment including heavy chain and light chain or again Chain variable region and light chain variable district.In a particular aspects, for the MEDI4736 in these methods provided herein or it is anti- Former binding fragment includes light chain variable district and weight chain variable district.In a particular aspects, in these methods provided herein MEDI4736 or its antigen-binding fragment include weight chain variable district and light chain variable district, wherein the weight chain variable district includes above CDR1, CDR2 and CDR3 sequence that shown Kabat is defined, and wherein the light chain variable district includes Karbate illustrated above CDR1, CDR2 and CDR3 sequence of definition.Those skilled in the art can easily identify well known by persons skilled in the art CDR definition that Chothia is defined, Abm is defined or other.In a particular aspects, for these methods provided herein In MEDI4736 or its antigen-binding fragment include such as resist in U.S. Patent number 8, the 2.14H9OPT disclosed in 779,108 The variable heavy chain and variable light CDR sequence of body, the patent are combined herein by quoting with its full text.

Anti- CTLA-4 antibody

The antibody for specifically binding CTLA-4 and suppressing CTLA-4 activity can be used for strengthening anti-tumor immune response.On with The information of Sibutramine Hydrochloride wood monoclonal antibody (or its antigen-binding fragment) in method provided herein can see (its of US 6,682,736 In it be referred to as 11.2.1), the disclosure content of the patent is combined herein by quoting with its full text.Sibutramine Hydrochloride wood monoclonal antibody is (also known as CP-675,206, CP-675, CP-675206 and for western wooden monoclonal antibody (ticilimumab)) be a kind of IgG₂Monoclonal resists Body, the monoclonal antibody have high selectivity for CTLA-4 and have blocked CTLA-4 and CD80's (B7.1) and CD86 (B7.2) With reference to.It has been demonstrated to cause ion vitro immunization to activate, and some patients treated with Sibutramine Hydrochloride wood monoclonal antibody have shown tumour and disappeared Move back.

Include heavy chain and light chain or weight chain variable district and light chain for the Sibutramine Hydrochloride wood monoclonal antibody in these methods provided herein Variable region.In a particular aspects, for the Sibutramine Hydrochloride wood monoclonal antibody in these methods provided herein or its antigen-binding fragment bag Light chain variable district and weight chain variable district are included, the light chain variable district includes amino acid sequence illustrated above, the weight chain variable district bag Include amino acid sequence illustrated above.In a particular aspects, for the Sibutramine Hydrochloride wood monoclonal antibody in these methods provided herein or Its antigen-binding fragment includes weight chain variable district and light chain variable district, and the wherein weight chain variable district includes Kabat illustrated above CDR1, CDR2 and CDR3 sequence of definition, and wherein the light chain variable district include Karbate illustrated above define CDR1, CDR2 and CDR3 sequences.Those skilled in the art can easily identify Chothia definition well known by persons skilled in the art , Abm is defined or other CDR definition.In a particular aspects, for the Sibutramine Hydrochloride wood monoclonal antibody in method provided herein or Its antigen-binding fragment is included such as the variable heavy chain and variable light CDR of the 11.2.1 antibody disclosed in US 6,682,736 Sequence, the patent are combined herein by quoting with its full text.

Other anti-CTLA-4 antibody are described in such as US 20070243184.In one embodiment, the anti-CTLA-4 Antibody is easy Puli's nurse agate, also referred to as MDX-010；BMS-734016.

OX40 activators

In by antigen elicitation procedure or after the initiation soon, OX40 activators and CD4⁺OX40 in T cell by Body phase interaction, so as to cause CD4⁺Response increase of the T cell to the antigen.Compared with the response to antigen alone, OX40 swashs Dynamic agent can increase T cell propagation with the OX40 acceptor interactions on antigentic specificity CD4+T cells.With OX40 activators not Existing situation is compared, and the elevated response to antigen can maintain the substantially longer period.Therefore, via OX40 excitements The stimulation of agent is identified come enhancement antigen specific immune response by strengthening the T cell of antigen such as tumour cell.OX40 excitements Agent is for example described in U.S. Patent number 6,312,700,7,504,101,7,622,444 and 7,959,925, and these are special Profit is combined herein by quoting with entire contents.Using the method for these activators for example in WO/2013/ in treatment of cancer It is described in 119202 and in WO/2013/130102, each in these patents is incorporated in by quoting with its full text This.

OX40 activators include but is not limited to OX40 binding molecules, such as Binding peptide, such as OX40 parts (" OX40L ") Or OX40 binding fragments, variant or derivatives thereof (the outer ligand domain of such as soluble cell and OX40L fusion proteins) and anti- OX40 antibody (for example, monoclonal antibody such as Humanized monoclonal antibodies) or its antigen-binding fragment, variant or derivative.It is anti- The example of OX40 monoclonal antibodies is for example described in U.S. Patent number 5,821,332 and 6,156,878, these patents Disclosure content is combined herein by quoting with entire contents.In certain embodiments, the anti-OX40 monoclonal antibodies are 9B12 Or its antigen-binding fragment, variant or derivative, such as Weinberg, A.D. et al., JImmunother [immunological therapy magazine] Described in 29,575-585 (2006), the document is combined herein by quoting with its full text.

In some aspects, present disclosure provide it is a kind of comprising the antibody VH and antibody VL anti-OX40 antibody of humanization or its resist Former binding fragment, the wherein VL include and reference amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ KPGKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK(SEQ ID NO：Or DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSKLHS GVPSRFSGSGSRT 22) DYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK(SEQ ID NO：23) have at least 70%, 75%, 80%, 85%th, the amino acid sequence of 90%, 95% or 100% uniformity

On the one hand, present disclosure offer is a kind of includes the antibody VH and antibody VL anti-OX40 antibody of humanization or its antigen knot Fragment is closed, the wherein VL includes amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLI YYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK(SEQ ID NO：22), and And the VH includes amino acid sequence QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGI T YHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS(SEQ ID NO： 24)。

In some aspects, present disclosure offer is a kind of includes heavy chain of antibody or its fragment and the people source of antibody light chain or its fragment Change anti-OX40 antibody or its antigen-binding fragment, the wherein heavy chain includes amino acid sequence

QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDT SKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：25), and the light chain includes amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKL LIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC(SEQ ID NO：26).

In other embodiments, it is specifically bound to OX40 antibody or its antigen-binding fragment is attached to and mAb 9B12 Identical OX40 epitopes.

By Morris et al., Mol Immunol [molecular immunology] .2007 Mays, 44 (12)：3112-3121 is to showing Example property humanization OX40 antibody is described, and the antibody has following sequence (SEQ ID NO：27)：

APLATDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKELLGGGSIKQIEDKIEEILSKIYHIENEIARIKKLIGERGHGGGSNSQVSHRYPRFQSIKVQFTEYKKEKGF ILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLN VTTDNTSLDDFHVNGGELILIHQNPGEFCVL

9B12 is muroid IgG1, for people OX40 (CD134) extracellular domain anti-OX40mAb (Weinberg, A.D. et al., J Immunother [immunological therapy magazine] 29,575-585 (2006)).Believe because 9B12 triggers for OX40 The ability of activator response of number conduction, stability and its produced by the high level of hybridoma, so from a series of anti- 9B12 is selected in OX40 monoclonal antibodies.For the use in clinical practice, by 9B12mAb phosphate buffered saline (PBS) (pH 7.0) balance, and adjusted its concentration to 5.0mg/ml by being percolated.

" OX40 parts " (" OX40L ") (is also referred to variously as Tumor necrosis factor ligand superfamily member 4, gp34, TAX Transcription activating glycoprotein 1 and CD252) antigen presenting cell (APC) is largely found in, and can activate B cell, dendritic cells (DC), Langerhans cell, be induced (Croft, M., (2010) on thick liquid cell DC and macrophage Ann Rev Immunol [immunology yearbook] 28：57-78).Other cells, including the T cell of activation, NK cells, hypertrophy are thin Born of the same parents, endothelial cell and smooth muscle cell can express OX40L in response to inflammatory cytokine (Id.).OX40L specificity knots Close OX40 acceptors.People's albumen is described in United States Patent (USP) 6,156,878.Mouse OX40L is in United States Patent (USP) 5,457,035 In be described.OX40L is expressed on the surface of cell, and including intracellular, cross-film and extracellular receptor binding domain. OX40L functional activity soluble form can be by producing, such as example in following patent in deletion cells with membrane spaning domain Described in：U.S. Patent number 5,457,035；6,312,700；6,156,878；6,242,566；6,528,055；6,528, 623；7,098,184；And 7,125,670, its disclosure content combines herein for all purposes.OX40L functional activity shape Formula is to retain the form for the ability for being specifically bound to OX40, the i.e. form with OX40 " receptor binding domain ".One reality Example is the amino acid 51 to 183 of human OX 40 L.Determine OX40L molecules or derivative be specifically bound to OX40 ability method Discussed below.Prepare and using OX40L and its derivative (derivative for such as including OX40L binding structural domains) Method is described in following patent：U.S. Patent number 6,156,878；6,242,566；6,528,055；6,528,623；7, 098,184；And 7,125,670, these patents also describe to include the protein for the OX40L soluble forms for being connected to other peptides, Such as human immunoglobulin(HIg) (" Ig ") Fc areas, these protein can produce the purifying to promote OX40 parts from culture cell, or Person's enhancing give in vivo to the stability of molecule after mammal (referring further to, U.S. Patent number 5,457,035 and 7,959, 925, both of which is combined herein by quoting with its full text).

As used herein, term " OX40L " includes whole OX40 parts, soluble OX40 parts and OX40 parts Functional activity part.Also included in OX40L definition is OX40 ligand variants, its amino acid sequence with it is naturally occurring OX40 ligand moleculars are different, but its reservation is specifically bound to the ability of OX40 acceptors.These variants in following patent Description：U.S. Patent number 5,457,035；6,156,878；6,242,566；6,528,055；6,528,623；7,098,184；With And 7,125,670.In the embodiment of correlation, present disclosure provides the OX40L's for the ability for having lost specific binding OX40 Phenylalanine at the position 180 of the receptor binding domains of mutant, such as amino acid 51 to 183, wherein human OX 40 L has been replaced For alanine (F180A).

OX40 activators include fusion protein, and in the fusion protein, OX40L one or more domains covalently connect It is connected to one or more other protein structure domains.The exemplary OX40L fusion proteins that can be used as OX40 activators are special in the U.S. It is described in profit number 6,312,700, the disclosure content of the patent is combined herein by quoting with its full text.In one embodiment In, OX40 activators include being self-assembled into the OX40L fusions of polymer (for example, tripolymer or six aggressiveness) OX40L fusion proteins Polypeptide.Such fusion protein is for example described in U.S. Patent number 7,959,925, and the patent is by quoting with its full text With reference to herein.Polymer OX40L fusion proteins should in the antigen specific immune of enhancing subject (especially people experimenter) Answer aspect and show increased effect, this is due to that it is spontaneously assemble into the ability of highly stable tripolymer and six aggressiveness.

In another embodiment, the OX40 activators that can be assembled into multimeric forms are included in N-terminal to C-terminal side The fused polypeptide of the following is included upwards：Immunoglobulin domains, the wherein immunoglobulin domains include Fc structures Domain, trimerising domain, the wherein trimerising domain include coiled coil trimerising domain；And acceptor integrated structure Domain, wherein this receptor binding structural domain are OX40 receptor binding domains, for example, OX40L or OX40 binding fragments, its variant or Derivative, the wherein fused polypeptide can be self-assembled into trimeric fusion albumen.In an aspect, polymer can be assembled into The OX40 activators of form can be attached to OX40 acceptors and stimulate the activity of at least one OX40 mediations.In some aspects, The OX40 activators include the extracellular domain of OX40 parts.

The trimerising domain that the OX40 activators of multimeric forms can be assembled into is used to promote single OX40L fusions more Peptide molecule is self-assembled into trimer protein.Therefore, the OX40L fused polypeptides with trimerising domain are self-assembled into tripolymer OX40L fusion proteins.In an aspect, the trimerising domain is isoleucine zipper motif domain (zipper domain) Or other coiled coil polypeptide structures.The trimerising domain of exemplary coiled coil includes：TRAF2(Step on Record Q12933, amino acid 299-348)；Platelet factor4 (accession number PO7996, amino acid 291-314)；Maternal egg - 4 (accession number O95460, amino acid 594-618) in vain；CMP (maternal albumen -1) (accession number NP-002370, amino acid 463- 496)；HSF1 (accession number AAX42211, amino acid/11 65-191)；And gulp down drink acceptor (Cubilin) (accession number NP- 001072, amino acid/11 04-138).In some particular aspects, trimerising domain includes TRAF2 trimerising domains, maternal Protein-4 trimerising domain or its combination.

MEDI6383 is to be specifically bound to people OX40 acceptors (TNFR family members) and trigger by people OX40 acceptors to believe Number conduction people's OX40 ligand i gG4P fusion proteins.MEDI6383 is made up of three different domains：(1) homologous three are formed Aggressiveness and the extracellular receptor binding domain of people's OX40 parts (RBD) for combining OX40 acceptors；(2) make that OX40 parts RBD's is same Source Trimeric structures it is stable from isoleucine zipper trimerising domain derived from TNFR correlation factors 2；And (3) human IgG 4 The crystallizable γ of fragment (Fc γ) domain, the domain promote Fc γ acceptor of the fusion protein when being attached to OX40 acceptors to gather Collection, and include serine in hinge area (IgG4P) and substitute to proline to lift two groups of OX40 part RBD homotrimers Stability.

In the particular embodiment, OX40 activators are modified to increase its serum half-life.For example, OX40 activators Serum half-life can be by being conjugated to increase with heterologous molecule such as seralbumin, antibody Fc district or PEG.In some embodiments In, OX40 activators can be conjugated to form immunoconjugates and/or fusion protein with other therapeutic agents or toxin.In some sides Face, OX40 activators can be prepared to help to give and to promote the stability of activating agent.

Antibody

Selective binding CTLA-4 and PD-L1 and suppress the antibody of CTLA-4 and PD-L1 combination or activation and can be used for The inventive method.Selective binding and to activate OX40 antibody in these methods of the present invention be useful.

In general, the preparation of antibody can be for example using traditional hybridoma technology (Kohler and Milstein (1975) Nature [nature], 256：495-499), recombinant DNA method (U.S. Patent number 4,816,567) or with antibody, text Storehouse carry out phage display (Clackson et al. (1991) Nature [nature], 352：624-628；Marks et al. (1991), J.Mol.Biol. [J. Mol. BioL], 222：581-597).For other antibody production techniques, referring further to Antibodies：ALaboratory Manual [antibody：Laboratory manual], editor Harlow et al., Cold Spring Harbor Laboratory [cold spring harbor laboratory], 1988.The invention is not restricted to any specific source, originating species, production Method.

Complete antibody, also known as immunoglobulin, typically tetramer glycosylated protein, the tetramer glycosylate egg White matter is made up of each about 25kDa two light (L) chain and each about 50kDa two heavy chains (H).It is designated as λ chains It is found in the two kinds of light chain of κ chains in antibody.According to the amino acid sequence of the constant domain of heavy chain, immune globulin Five major classes can be divided into vain：A, D, E, G and M, and some can be further divided into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The subunit structure and 3-d modelling of different classes of immunoglobulin are well known in the present art.For antibody knot The summary of structure, referring to Lip river (Harlow) et al. is breathed out, see above.Briefly, each light chain is by a N- terminal variable domain (VL) formed with a constant domain (CL).Each heavy chain is by a N- terminal variable domain (VH), three or four perseverances Constant domain (CH) and hinge area are formed.Closest VH CH domains are designated as CH1.VH and VL domains are by being referred to as framework Four regions composition of the relative conserved sequence in area (FR1, FR2, FR3 and FR4), these framework regions determine to be referred to as complementarity Three regions of the hypervariable sequence in area (CDR) form a support.CDR includes most of be responsible for and antigentic specificity interaction Residue.Three CDR are referred to as CDR1, CDR2 and CDR3.CDR components on heavy chain are referred to as H1, H2 and H3, and on light chain CDR components are accordingly called L1, L2 and L3.CDR3 and particularly H3, it is that molecular diversity is most in antigen-binding domains Big source.For example, H3 can be as short as two amino acid residues or more than 26 amino acid residues.

Fab fragments (antigen-binding fragment) are by the VH-CH1 and VL-CL by the disulfide bond covalent attachment between constant region Domain forms.In order to which what is dissociated when overcoming VH the and VL domains being not covalently linked in Fv to be co-expressed in host cell inclines To so-called single-stranded (sc) Fv fragment (scFv) can be built.In scFv, flexible and sufficiently long polypeptide is last by VH C End links to VL N- ends or VL C-terminal is linked to VH N- ends.Most generally, 15 residue (Gly4Ser) 3 peptides (SEQ ID NO：28) it can be used as joint, but other joints are also known in the art.

Antibody diversity is the result of the combination assembling of multiple germ line genes of encoding variable regions and various somatic events. The restructuring that somatic events include variable gene segment and connection (J) constant gene segment C with diversity (D) is complete to form one The restructuring of Zheng VH areas and variable gene segment and connection constant gene segment C forms a complete VL area.Regrouping process is in itself It is inaccurate, causes the missing in the amino acid in V (D) J crosspoints or addition.These multifarious mechanism occur sudden and violent in antigen In the B cell of development before dew.After antigenic stimulus, the antibody gene expressed in B cell undergoes somatic mutation.

The number of estimation based on germline gene segment, the random restructuring of these sections and random VH-VL pairings, can be produced Up to 1.6 × 10⁷Individual different antibody (Fundamental Immunology [basic immunology], the 3rd edition, Paul writes, Raven Press [Lei Wen publishing houses], New York, NY, 1993).When it will promote antibody diversity (such as somatic mutation) When other method is taken into account, it is believed that can potentially produce more than 1 × 10¹⁰Individual different antibody (Immunoglobulin Genes [immunoglobulin gene], second edition, editor Jonio et al., Academic Press, San Diego, Calif. [add The academic press in Li Funiya states Santiago], nineteen ninety-five).Because many methods are related to antibody diversity, independently produce Antibody will be extremely impossible with identical or even essentially similar amino acid sequence in multiple CDR.

It there is provided herein exemplary anti-CTLA-4 and anti-PD-L1CDR sequence.Structure for carrying CDR will substantially On be a heavy chain of antibody or light chain or one part, the wherein CDR is positioned corresponding to naturally occurring VH and VL CDR position Put.Structure and the position in immunoglobulin variable domain domain can be determined, for example, Kabat et al. is such as described in, Sequences Of Proteins of Immunological Interest [immunology protein sequence interested], No. 91-3242, National Institutes of Health Publications, Bethesda, Md. [Maryland State Bei Saisida states Vertical Institutes of Health Research publication], 1991.

The antibody (for example, anti-CTLA-4, anti-PD-L1, anti-OX40) of the present invention can optionally include antibody constant region Or part thereof.For example, VL domains can have the antibody light chain constant domain in the attachment of its C- end, including people C κ or C λ Chain.Similarly, the specific antigen-binding domains based on VH domains can have one heavy chain immunoglobulin of attachment All or part of, the heavy chain immunoglobulin originates from any antibody isotope, such as IgG, IgA, IgE and IgM, and any The isotope subclass, it includes but is not limited to IgG1 and IgG4.

One of ordinary skill in the art is it will be recognized that the antibody of the present invention can be used for detecting, measure and suppressing and CTLA- Protein somewhat different 4 and PD-L1.These antibody are estimated to be expected to keep the specificity combined, if target protein include with Any sequence (as described herein) of at least 100,80,60,40 or 20 continuous amino acids have at least about 60%, 70%, 80%th, the sequence of 90%, 95% or higher uniformity.Percent Identity is determined by standard alignment algorithms, these marks Quasi- alignment algorithm such as Altshul et al. (1990, J.Mol.Biol. [J. Mol. BioLs], 215：403-410) describe Basic Local Alignment Tool (BLAST), Needleman et al. (1970, J. Mol. BioL, 48：Calculation 444-453) Method, or Meyer this (Meyers) et al. (1988, Comput.Appl.Biosci. [computer application bioscience], 4：11-17) Algorithm.

Except sequence homology analysis, epitope mapping can be carried out (see, e.g., Epitope Mapping Protocols [Epitope mapping experiments scheme], edit Morris, Humana Press [Hu Mana publishing houses], 1996) and two level Analyzed with tertiary structure to identify the compound by the specific 3D structures of disclosed antibody hypothesis and they and antigen.These sides Method includes but is not limited to：X-ray crystallography (Engstom (1974) Biochem.Exp.Biol. [Biochemistry Experiment biologies Learn], 11：7-13) and present disclosure antibody virtual representation microcomputer modelling (Fletterick et al. (1986) Computer Graphics and Molecular Modeling [computer graphics and molecule modeling], in Current In Communications in Molecular Biology [Current Protocols communication], Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. [cold spring harbor laboratory, New York Cold SpringHarbor]).

Derivative

The antibody (such as anti-CTLA-4, anti-PD-L1, anti-OX40) of the present invention can include the variant of these sequences, its Remain the ability for specifically binding their target.These variants can be by those skilled in the art by using in this area Derived from sequence of the known technology from these antibody.For example, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can be manufactured in FR and/or CDR, is lacked Lose or add.Although FR change is typically designed to improve the stability and immunogenicity of antibody, and CDR change is typical Ground is designed to increase affinity of the antibody to its target.It is of the same race different that FR variant also includes naturally occurring immunoglobulin Type.The conventional skill for the affinity antibody that the increased change of these affinity can change CDR and test its target empirically by being related to Art determines.For example, conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can be manufactured in any one in disclosed CDR.Can basis Antibody Engineering [antibody engineering], second edition, Oxford University Press, are edited in Borrebaeck, nineteen ninety-five The method of description carries out various changes.These include but is not limited to by the functionally equivalent amino acid residue of sequence interior coding The nucleotide sequence that the substitution of different codons changes, so as to produce the change of " silence ".For example, nonpolar amino acid includes third Propylhomoserin, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.Polar neutral amino acid bag Include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.Positively charged (alkalescence) Amino acid includes arginine, lysine and histidine.Negatively charged (acidity) amino acid includes aspartic acid and glutamic acid.

The derivative and analog of the antibody of the present invention can be produced by various technologies as known in the art, these Technology includes restructuring and synthesis method (Maniatis (1990) Molecular Cloning, A Laboratory Manual [molecular cloning, laboratory manual], second edition, cold spring harbor laboratory, New York Cold SpringHarbor；With Bodansky et al. (1995) The Practice of Peptide Synthesis [putting into practice for peptide symthesis], second edition, Springer Verlag, moral State Berlin).

In one embodiment, for the amino acid sequence variation that is fabricated to VH domains of the present invention VH domains side Method, which is included in the amino acid sequence of presently disclosed VH domains, adds, lacks, substitutes or inserts one or more amino acid A step, optionally by the VH domains so provided and one or more VL domain combinations, and test the VH structures Domain or VH/VL combinations or the combination of molecule of the antigen binding.Similar approach can be used, wherein by VL domains disclosed here One or more sequence variants and one or more VH domain combinations.

By Stemmer (Nature [nature] (1994) 370：Similar reorganization or combination technique 389-391) are also disclosed, Stemmer describe with the technology of beta-lactamase gene-correlation but observe this method can be used for produce antibody.

In a further embodiment, people can use the random of the VH and/or VL genes of one or more selections to lure Become the novel VH or VL areas for producing and carrying the one or more sequences for being derived from sequence disclosed herein.Gram et al. (Proc.Nat.Acad.Sci.U.S.A. [NAS's proceeding] (1992) 89：3576-3580) describe it is a kind of this The technology of sample, fallibility PCR.

Another workable method is to guide mutagenesis to the CDR of VH or VL genes.Barbas et al. (Proc.Nat.Acad.Sci.U.S.A. [NAS's proceeding] (1994) 91：3809-3813) and Schier et al. (J.Mol.Biol. [J. Mol. BioL] (1996) 263：551-567) disclose these technologies.

Similarly, one or more or all three CDR can be bonded in the pedigree of VH or VL domains, be then directed to The antigen-binding fragment special to CTLA-4 or PD-L1 screens to these domains.

The part in immunoglobulin variable domain domain by including generally as at least one in CDR set forth herein, And optionally, carry out the intervention framework region of scFv fragments set forth herein freely.The part can include one in FR1 and FR4 It is individual or two at least about 50%, this 50% be FRI C- ends 50% and FR4 N- ends 50%.The reality of variable domains The N- ends of matter part or the other residue of C- extreme ends can be generally not with naturally occurring variable domains region Those associated residues.For example, by recombinant DNA technology build antibody can cause the N- that is encoded by introduced joint or The introducing of C- terminal residues, to promote clone or other maneuvering sequences.Other maneuvering sequences include introducing joint so as to can Structure changes domain is joined to other protein sequence, these protein sequences include immunoglobulin heavy chain constant region, other can Structure changes domain (such as in generation of double antibody) or protein labeling, as discussing in further detail below.

Skilled artisan will recognize that antibody of the invention can include antigen-binding fragment, the antigen binding fragment Section only includes the single CDR from VL or VH domains.Any one in these single-stranded specific binding domains can be used for Screening can form the complementary domain of a bi-domain specific antigen binding fragment, and the antigen-binding fragment can be such as It is attached to CTLA-4 and PD-L1.

Antibody (for example, anti-CTLA-4 and/or anti-PD-L1) of the invention described herein may be connected to another feature Molecule, such as another peptide or protein matter (albumin, another antibody etc.).For example, these antibody can pass through chemical crosslinking Or connected by recombination method.These antibody can also be connected to a variety of nonproteins in the way of listed in following patent and gather Compound is for example, one kind in polyethylene glycol, polypropylene glycol or polyoxyalkylene：U.S. Patent number 4,640,835；4,496,689；4, 301,144；4,670,417；4,791,192；Or 4,179,337.Antibody can be changed by covalently conjugated to polymer Modification is learned, such as increases its circulating half-life.Be attached these antibody exemplary polymer and method also in U.S. Patent number 4, 766,106；4,179,337；4,495,285, and 4,609,546.

Disclosed antibody can also change with the glycosylation pattern different from native pattern.For example, one or more Individual carbohydrate portions can be deleted and/or one or more glycosylation sites can be added to original antibodies.By sugar Base site is added to presently disclosed antibody can be by changing amino acid sequence with comprising glycosylation as known in the art Site consensus sequence is realized.On antibody increase carbohydrate portions quantity another way be by make glucosides with The amino acid residue of antibody carries out chemistry or enzymatic of glucosides.These methods are described in WO 87/05330 and in Aplin et al. (1981) CRC Crit.Rev.Biochem. [CRC biochemistries critical review], 22：In 259-306.By any carbon hydrate Thing part removes from antibody and can realized by chemistry or enzymatic method, for example, such as Hakimuddin et al. (1987), Arch.Biochem.Biophys. [biochemistry and biophysics collected papers], 259：52；And Edge et al. (1981) Anal.Biochem. [analytical biochemistry], 118：131；And pass through Thotakura et al. (1987) Meth.Enzymol. [Enzymology method], 138：Described by 350.Antibody can also be labelled with detectable or functional label.Detectable label bag Radioactive label is included, such as 131I or 99Tc, can be used conventional chemical that they are attached into antibody.Detectable label also includes enzyme Mark, such as horseradish peroxidase or alkaline phosphatase.Detectable label further comprises chemical part (such as biotin), and it can Detected via specific homologous detectable part (such as avidin of mark) is bound to.

Cover CDR sequence antibody different from the antibody illustrated at this only on non-intrinsically safe within the scope of the invention. Typically, an amino acid is substituted by the related amino acid with similar electric charge, hydrophobicity or stereochemical characteristics.These substitutions By within the common skill of technical staff.Unlike more substantial changes in CDR, can be carried out in FR, without right The binding property of antibody adversely affects.Change to FR include but is not limited to derived from humanizing non-human or engineering for Antigen contact is important some Framework residues for stable bond site, for example, changing the class of constant region or subclass, changing Become can change effector function (such as Fc acceptors combination) particular amino acid residue (for example, if description is in U.S. Patent number 5, 624,821 and 5,648,260；And Lund et al. (1991) J.Immun [Journal of Immunology] .147：2657-2662 and Morgan et al. (1995) Immunology [immunology] 86：In 319-324) or change constant region derived from species.

It will be apparent to one skilled in the art that above-described change is not all of in detail, and due to present disclosure Teach, many other changes will be obvious to those skilled in the art.

Combination treatment

Use the combination of the present invention, anti-CTLA-4 antibody, anti-PD-L1 antibody or its antigen binding fragment as provided here Section and OX40 activators or its antigen-binding fragment can cause additive effect or collaboration effect to treat the patient with solid tumor Should.As used herein, term " collaboration " refers to combination treatment (for example, anti-CTLA-4 antibody, anti-PD-L1 antibody or its antigen The combination of binding fragment and OX40 ligand fusion proteins).

Combination treatment is (for example, anti-CTLA-4 antibody, anti-PD-L1 antibody or its antigen-binding fragment and OX40 parts melt The combination of hop protein) cooperative effect allow using lower dosage one or more therapeutic agents and/or to solid tumor Patient gives the therapeutic agent with smaller frequency.The treatment is given using the therapeutic agent of lower dosage and/or with less frequency The ability of method is reduced gives the related toxicity of the therapy without the reduction therapy in the treatment of solid tumor with to subject The effect of.In addition, the effect of cooperative effect can cause therapeutic agent to be improved in the management of solid tumor, treatment or in improving.Treatment The cooperative effect of the combination of agent can avoid or reduce the bad or unwanted side effect related to using any monotherapy.

In combination treatment, anti-CTLA-4 antibody, anti-PD-L1 antibody or its antigen-binding fragment and OX40 parts melt The combination of hop protein can be optionally included in identical pharmaceutical composition, or can be included in one or more independent Pharmaceutical composition in.In some aspects, according to the Pharmaceutical composition of present disclosure include it is pharmaceutically acceptable, avirulent, Sterile carrier, such as physiological saline, avirulent buffer, preservative.For what is used in treatment method disclosed here Suitable formulations are for example, Remington ' s Pharmaceutical Sciences [Lei Mingdunshi pharmaceutical sciences]) Mack Publishing Co. [Mack publishing company], it is described in the 16th edition (1980).

Kit

The invention provides the kit for strengthening antitumor activity.In one embodiment, the kit includes containing There are the Therapeutic combinations of anti-CTLA-4 antibody, anti-PD-L1 antibody and OX40 activators (for example, OX40 ligand fusion proteins) Thing.

In certain embodiments, the kit includes a sterile chamber for including therapeutic composition；Such container can To be box, ampoule, bottle, bottle, pipe, sack, pouch, blister package or other suitable containers known in the art Form.Such container can be made up of plastics, glass, laminated paper, metal foil or the other materials suitable for accommodating medicine.

If desired, the kit further comprises the specification of the therapeutic combination for giving the present invention.Having In body embodiment, these specifications include at least one of the following：The description of therapeutic agent；For strengthening antitumor activity Dosage schedule and give；Points for attention；Warning message；Indication；Contraindication；Overdose information；Adverse reaction；Animal drugs It is of science；Clinical research；And/or bibliography.These specifications can be directly printed upon on container (when it is present), or as mark Label are applied to container, or as single page, pamphlet, card or the file provided in a reservoir or together.

Except as otherwise noted, implementation of the invention uses is within the scope of the experience of those skilled in the art well Molecular biology (including recombinant technique), microbiology, cell biology, biochemistry and immunologic routine techniques.These Technology has been fully explained in the literature, such as " Molecular Cloning：A Laboratory Manual [molecular clonings： Laboratory manual] ", second edition (Sambrook, 1989)；" Oligonucleotide Synthesis [oligonucleotide synthesis] " (Gait, 1984)；" Animal Cell Culture [animal cell culture] " (Freshney, 1987)；“Methods in Enzymology [Enzymology method] ", " Handbook of Experimental Immunology [experiment immunization learns to do volume] " (Weir, 1996)；" Gene Transfer Vectors for Mammalian Cells [are used for the gene of mammalian cell Transfer vector] " (Miller and Calos, 1987)；" [current point of Current Protocols in Molecular Biology Sub- biological method] " (Ausubel, 1987)；“PCR：The Polymerase Chain Reaction[PCR：Polymerase chain Formula is reacted] " (Mullis, 1994)；" Current Protocols in Immunology [immunology modern experimental scheme] " (Coligan, 1991).These technologies are applied to the production of the polynucleotides and polypeptide of the present invention, and according to so, can be by Consider to be used to make and implement the present invention.The particularly useful technology of specific embodiment will be discussed in following part.

Provide following example and be in order to provided to those of ordinary skill in the art how to prepare and using measure, screening and One of the treatment method of the present invention is complete described and illustrated, without being intended to limit inventors believe that being the hair of oneself Bright scope.

Example

The combination of example 1.OX40 ligand fusion proteins, anti-CTLA-4 antibody and anti-PD-L1 antibody suppresses homology model The growth of middle cancerous cell line

In MCA205, the homologous sarcoma model of mouse, by mOX40L FP (mouse OX40 ligand fusion proteins), anti-PD- L1 (10F.9G2) and anti-CTLA-4 (9D9) antitumor activity as monotherapy, as double combinations therapy or conduct Triple combination treatments are assessed.Compared to giving control sample or give any of single above-mentioned medicament or with dual group Conjunction is given, and mOX40L FP and 10F.9G2 and 9D9 combination, which is given, causes higher antitumor activity.

Test sample is obtained as below：Anti- CTLA-4 (9D9, BioXcell company, western Lebanon, the state of New Hampshire)； Anti- PD-L1 (10F.9G2, BioXcell company, western Lebanon, the state of New Hampshire)；(mouse OX40L is merged with OX40L FP Albumen, Med Muniz Co., Ltd (MedImmune), Gaithersburg, the Maryland State).MEDI6383, MEDI4736 and Sibutramine Hydrochloride Wooden monoclonal antibody difference nonrecognition mouse OX40, PD-L1 or CTLA-4.Generate muroid OX40 ligand i gG1 fusion proteins (mOX40L FP), the fusion protein combination mouse OX40, triggering OX40 carry out signal transduction and are used as MEDI6383 replacement mouse OX40 activators.10F.9G2 is the commercially available rat IgG2b antibody for mouse PD-L1；And 9D9 is to be directed to mouse CTLA-4 commercially available mouse IgG 2b antibody.Therefore, it is contemplated that effect and/or activity correspond to effect in the mankind and/or Activity (for example, when using human antibody, amino acid sequence etc.).Control sample is obtained as below：OX40L FP Y182A (have Y To the mouse OX40 ligand fusion proteins of A 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, Med Muniz Co., Ltd, Gaithersburg, the Maryland State)；It is small Mouse IgG2b isotype controls (MPC-11, BioXcell company, western Lebanon, the state of New Hampshire)；It is of the same race with rat IgG2b Type compares (MPC-11, BioXcell company, western Lebanon, the state of New Hampshire).Y182A mutant mice OX40L mouse The control of IgG1 fusion proteins is included in receptor binding domain at position 182, and there are single amino acids to be mutated (Y to A amino Acid change) mOX40L FP, the mOX40L FP prevent mOX40L combination mouse OX40, but do not influence the overall of mOX40L and tie Structure.OX40L FP Y182A do not combine natural mouse or people OX40, and therefore serve as OX40L：OX40 interacts Negative control.

The homologous tumours of following MCA205 are established in C57BL/6 mouse.MCA205 cells are derived from Providence cancer Center (Providence Cancer Center) (Portland, Oregon), and make it in the culture medium (Lip rivers of RPMI 1640 This dimension park memorial institute (Roswell Park Memorial Institute) 1640 culture medium, Life Technologies, Inc. (Life Technologies), Carlsbad, California) in grow, 1640 culture medium is supplemented with 10% tire ox Serum (Life Technologies, Inc., Carlsbad, California).MCA205 is that the mouse soft tissue sarcoma of chemical induction swells Oncocyte.By will be suspended in 0.1mL phosphate buffered saline (PBS)s 2.5 × 10⁵Under MCA205 cell skins (SC) be injected to 7 to The right side flaps of 9 week old female C57BL/6 mouse (Ha Lan Laboratories, Incs, Indianapolis, the state of Indiana) is established Allograft.C57BL/6 (altogether 108) female mices are studied for this.11 days after transplanting, grown in tumour each Group average external volume is 185mm³±1mm³C57BL/6 mouse are randomly assigned afterwards.By group name, animal numbers, dosage level and agent Amount timetable is presented in table 1.

The research and design of table 1：MCA205 homology models

F=females；Isotype controls mixture contains the clone of the Fc domains with mIgG2b, rIgG2b and mIgG1 respectively MPC-11, LTF2 and mOX40L FP Y182A；IP=intraperitoneals；M/F=males/female；MAb=monoclonal antibodies； MOX40L FP=muroid OX40 part muroid IgG1 fusion proteins；NA=is unavailable, because these animal untreateds；ROA= Give approach.

^aDose volume：0.2mL.

All test samples and control sample are given by (IP) injection of intraperitoneal.There is no animal replacement.Face for bad Bed body sign monitors and monitors the general health of mouse every two weeks for body weight daily.If it is noted that hindlimb paralysis, breathing Distress, 20% body weight loss, gross tumor volume are more than 2000mm³Fester or necrosis tumour, immediately by using CO₂Asphyxia is human Ground is put to death by animal.All experiments are carried out to enter street crossing according to AAALAC and Med Muniz Co., Ltd's IACUC guides Treatment and Institutional Animal Care.

Kind of calliper tumour is used three-times-weekly or twice, and uses below equation：Gross tumor volume=[length (mm) × Width (mm)²]/2 (wherein length is defined as longer side, and width is defined as perpendicular to the shorter side of length) are come Calculate gross tumor volume.The antitumor action each organized is represented as Tumor growth inhibition (TGI), and it is calculated as below：Percentage TGI =(1-T/C) × 100, wherein, T=final doses are later from the final gross tumor volume for the treatment of group, and C=final doses are later From the final gross tumor volume of control group.If not having measurable tumour after treatment, tumour growth response is classified as completely Response (CR).

The one-factor analysis of variance (One-way ANOVA) is used for determining mean tumour volume difference.Tested in significant F In the case of, test (where applicable) using Deng Nite (Dunnett ' s) or Sidak ' s Multiple range tests.Where applicable, to tumour body Product is converted using log10 and explains heteroscedasticity.P value ＜ 0.05 is considered as significant.Use Log-Rank Test (logrank Test the paired survival curve of comparison) is carried out.By Bonferroni correct applications in Multiple range test to control Family-wise error rate (familywise error rate).Prism 6.03 for Windows is used for being analyzed.P values ＜ 0.05 (is not adjusted ) it is considered as significant.

Compared to untreated and isotype controls group mouse, with mOX40L FP, anti-CTLA-4mAb and anti-PD-L1 MAb treats mouse group as single medicament, causes the growth of MCA205 tumour cells to reduce (table 2, Figure 1A).Compared to each Kind individually corresponds to medicament, and carrying out treatment to mouse with anti-CTLA-4mAb and anti-PD-L1 mAb combination causes MCA205 to swell Horizontal similar (table 2, Figure 1A) of the tumour growth of oncocyte.Compared to single anti-PD-L1 mAb and single anti-CTLA-4 MAb or compared to control group but not compared with single mOX40L FP, with mOX40L FP and anti-PD-L1 mAb or anti- CTLA-4 mAb combination, which carries out treatment to mouse, makes the growth of MCA205 tumour cells reduce (table 2, Figure 1A).Compared to control The combination of group, individually every kind of therapeutic agent or any two in these medicaments, with mOX40L FP and anti-PD-L1 mAb and Anti- CTLA-4mAb combination, which carries out treatment to mouse, makes the growth of MCA205 tumour cells reduce (table 2, Figure 1A).

Treatment group of the table 2 in MCA205 homology models, the number in the percentage TGI of the 25th day and complete response person

IP=intraperitoneals；NA=is disabled；TGI=Tumor growth inhibitions；V=volumes.

^aN=11

^bFor mOX40L FP, all animals received 200 μ L test sample IP at the 11st and 15 day, and for anti-CTLA- 4mAb and anti-PD-L1 mAb, all animals received 200 μ L test samples IP at the 11st, 15,18 and 22 day.

^c%TGI=[1- (the average tumor V for the treatment of group) ÷ (the average tumor V of isotype controls group)] × 100

^dThere is the number that gross tumor volume measurement is registered as the animal of 0 group at the end of research.

Observe that the reaction between each animal is heterogeneous generally in homology model.However, when for combination treatment, Response increase to mOX40L FP, anti-CTLA-4 mAb and anti-PD-L1 mAb (is schemed from each animal tumor growth curve chart It is obvious in 1B-1D).Due to big tumor size, at the 45th day to untreated and isotype controls treatment dynamic Thing implements euthanasia (Figure 1B).

Relative to the treatment carried out by the use of single antibody as monotherapy under identical dosage level, anti-CTLA-4 MAb and anti-PD-L1 mAb combination is given (such as to be defined without result in antitumor activity increase by percentage TGI and CR number ) (table 2, Fig. 1 C).Controlled with 0 in 11 mouse being treated with anti-CTLA-4 mAb and with single anti-PD-L1 mAb 1 in 11 mouse treated is compared, and complete response (table is all not observed in the mouse of 11 in the group of combination treatment 2)。

It is anti-relative to the treatment carried out by the use of single mOX40L FP as monotherapy under same dose level CTLA-4 mAb give to mOX40L FP combination causes similar antitumor activity (such as by similar percentage TGI and CR What number defined) (table 2, Fig. 1 D)；Relative to by the use of single anti-CTLA-4 mAb as monotherapy under same dose level The treatment of progress, cause higher antitumor activity (as defined by percentage TGI and CR number) (table 2, Fig. 1 D).With with 0 in 11 mouse of anti-CTLA-4 mAb treatment and 2 phases in 11 mouse through single mOX40L FP treatments Than there is 5 to observe complete response (table 2) in 11 mouse in the group of combination treatment.

It is in relative to by the use of single medicament as monotherapy or dual therapy combination every kind of under same dose level The treatment of progress, anti-CTLA-4 mAb and anti-PD-L1 mAb give with mOX40L FP combination together causes antitumor activity Increase by higher TGI and greater number CR (as defined) (table 2, Fig. 1 D).With with anti-CTLA-4/PD-L1 mAb groups Close 0 in 11 mouse for the treatment of, 5 in 11 mouse through anti-CTLA-4/mOX40L FP combined therapies and pass through 3 in 11 mouse of anti-PD-L1/mOX40L FP combined therapies are compared, in the mouse of 11 in the group of three recombinations There are 8 to observe complete response (table 2).

The mouse of untreated gives isotype controls or anti-CTLA-4 mAb mouse and does not survive to research and tie The middle position time-to-live of beam, wherein mouse is 23 days, 23 days and 29 days (Fig. 1 E) respectively.For every kind of medicament, anti-PD-L1 Giving for mAb or mOX40L FP causes the middle position time-to-live to increase to 31 days.Anti- PD-L1 mAb and mOX40L are being given respectively After FP, 1 in 11 mouse and 2 survivals in 11 mouse terminate (at the 70th day) (Fig. 1 E) to research.

When being used as monotherapy under same dose level, relative to the treatment carried out with single any antibody, Anti- CTLA-4 mAb and anti-PD-L1 mAb combination is given increases (Fig. 1 F) without result in activity.When with 29 days (anti-CTLA- 4 mAb) with when (anti-PD-L1 mAb) is compared within 31 days, the middle position time-to-live is 31 days.There is no animal survival to terminate to the research.

When being used as monotherapy under same dose level, relative to the treatment carried out with single any antibody, Anti- CTLA-4 mAb and mOX40L FP combination, which is given, causes activity increase (Fig. 1 F).When with 31 days (anti-PD-L1 mAb) and When 31 days (mOX40L FP) is compared, the middle position time-to-live is 45 days.In addition, work as and 1 (anti-PD-L1 in 11 mouse When mAb) being compared with 2 (mOX40L FP) in 11 mouse, 11 in the group of anti-PD-L1 mAb/mOX40L FP combinations There are 3 survivals to terminate to research in mouse.

Relative to what is carried out with single any medicament or with the double combinations of these medicaments under same dose level Treatment, anti-CTLA-4 and anti-PD-L1 mAb and mOX40L FP combination, which is given, causes activity increase (Fig. 1 F).Due to survival The animal numbers terminated to research, the group calculating middle position time-to-live for three recombinations is impossible.In addition, when with 0 (p=0.0002 in 11 mouse of anti-CTLA-4/PD-L1 mAb combined therapies；Significantly), with anti-CTLA-4/ 5 (p ＞ 0.05 in 11 mouse of mOX40L FP combined therapies；It is inapparent) and with anti-PD-L1/mOX40L FP 3 (p=0.21 in 11 mouse of combined therapy；It is inapparent) compare (using the Bonferonni for Multiple range test Adjustment, is compared survival curve in pairs) when, there are in 11 mouse in the group of three recombinations 8 survivals to be tied to research Beam (Fig. 1 F).

Other embodiment

In from the foregoing description, it will be apparent that, invention described herein can be made change and change so that its It is adapted to various uses and situation.These embodiments are also within the scope of the following claims.

Narration in any definition of this variable to key element inventory includes the variable-definition being any single key element Or the combination (or secondary combination) of listed elements.The narration of embodiment herein include as any single embodiment or with it is any The embodiment that other embodiment or part thereof combines.

Whole patents for being referred in this specification and publication by quote with such as every part of single patent and publication Thing is pointed out specifically and individually to be incorporated by reference identical degree and combined herein.

Sequence table

SEQ ID NO：1MEDI4736 VL

＞ PCT/US2010/058007_77 come from PCT/US2010/058007 organisms：The sequence of homo sapiens 77EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK

SEQ ID NO：2MEDI4736 VH

＞ PCT/US2010/058007_72 come from PCT/US2010/058007 organisms：The sequence of homo sapiens 72EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS

SEQ ID NO：3-MEDI4736 VH CDR1

＞ PCT/US2010/058007_73 come from PCT/US2010/058007 organisms：The sequence 73RYWMS of homo sapiens

SEQ ID NO：4-MEDI4736 VH CDR2

＞ PCT/US2010/058007_74 come from PCT/US2010/058007 organisms：The sequence of homo sapiens 74NIKQDGSEKYYVDSVKG

SEQ ID NO：5-MEDI4736 VH CDR3

＞ PCT/US2010/058007_75 come from PCT/US2010/058007 organisms：The sequence of homo sapiens 75EGGWFGELAFDY

SEQ ID NO：6-MEDI4736 VL CDR1

＞ PCT/US2010/058007_78 come from PCT/US2010/058007 organisms：The sequence of homo sapiens 78RASQRVSSSYLA

SEQ ID NO：7-MEDI4736 VL CDR2

＞ PCT/US2010/058007_79 come from PCT/US2010/058007 organisms：The sequence 79DASSRAT of homo sapiens

SEQ ID NO：8-MEDI4736 VL CDR3

＞ PCT/US2010/058007_80 come from PCT/US2010/058007 organisms：The sequence 80QQYGSLPWT of homo sapiens

SEQ ID NO：9 Sibutramine Hydrochloride wood monoclonal antibodies

＞ SEQ ID NO：22 come from US 6,682,736

PSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

SEQ ID NO：10 Sibutramine Hydrochloride wood monoclonal antibody VH

＞ SEQ ID NO：9 come from US 6,682,736

GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVH

SEQ ID NO：11- Sibutramine Hydrochloride wood monoclonal antibody VH CDR1

GFTFSSYGMH

SEQ ID NO：12- Sibutramine Hydrochloride wood monoclonal antibody VH CDR2

VIWYDGSNKYYADSV

SEQ ID NO：13- Sibutramine Hydrochloride wood monoclonal antibody VH CDR3

TAVYYCARDPRGATLYYYYYGMDV

SEQ ID NO：14- Sibutramine Hydrochloride wood monoclonal antibody VL CDR1

RASQSINSYLD

SEQ ID NO：15- Sibutramine Hydrochloride wood monoclonal antibody VL CDR2

AASSLQS

SEQ ID NO：16- Sibutramine Hydrochloride wood monoclonal antibody VL CDR3

QQYYSTPFT

SEQ ID NO：17-MEDI6383 OX40 activators

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKDQDKIEALSSKVQQLERSIGLKDLAMADLEQKVLEMEASTQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI MKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDD FHVNGGELILIHQNPGEFCVL

Sequence table

<110>Med Muniz Co., Ltd

<120>For treating the therapeutic combination and method of neoplasia

<130> BTO-200WO1

<140>

<141>

<150> 62/167,625

<151> 2015-05-28

<160> 28

<170>PatentIn version 3s .5

<210> 1

<211> 108

<212> PRT

<213>Homo sapiens

<400> 1

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 2

<211> 121

<212> PRT

<213>Homo sapiens

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 5

<212> PRT

<213>Homo sapiens

<400> 3

Arg Tyr Trp Met Ser

1 5

<210> 4

<211> 17

<212> PRT

<213>Homo sapiens

<400> 4

Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210> 5

<211> 12

<212> PRT

<213>Homo sapiens

<400> 5

Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr

1 5 10

<210> 6

<211> 12

<212> PRT

<213>Homo sapiens

<400> 6

Arg Ala Ser Gln Arg Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 7

<211> 7

<212> PRT

<213>Homo sapiens

<400> 7

Asp Ala Ser Ser Arg Ala Thr

1 5

<210> 8

<211> 9

<212> PRT

<213>Homo sapiens

<400> 8

Gln Gln Tyr Gly Ser Leu Pro Trp Thr

1 5

<210> 9

<211> 139

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 9

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

1 5 10 15

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asp Trp Tyr Gln Gln Lys

20 25 30

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

35 40 45

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

50 55 60

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

65 70 75 80

Cys Gln Gln Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys

85 90 95

Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

100 105 110

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

115 120 125

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135

<210> 10

<211> 167

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 10

Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser

1 5 10 15

Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro

20 25 30

Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn

35 40 45

Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

50 55 60

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

65 70 75 80

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Arg Gly Ala Thr Leu

85 90 95

Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His

165

<210> 11

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 11

Gly Phe Thr Phe Ser Ser Tyr Gly Met His

1 5 10

<210> 12

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 12

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

1 5 10 15

<210> 13

<211> 16

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 13

Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 14

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 14

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asp

1 5 10

<210> 15

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 15

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 16

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 16

Gln Gln Tyr Tyr Ser Thr Pro Phe Thr

1 5

<210> 17

<211> 402

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 17

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Asp Gln Asp Lys Ile Glu Ala Leu Ser Ser Lys

225 230 235 240

Val Gln Gln Leu Glu Arg Ser Ile Gly Leu Lys Asp Leu Ala Met Ala

245 250 255

Asp Leu Glu Gln Lys Val Leu Glu Met Glu Ala Ser Thr Gln Val Ser

260 265 270

His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr

275 280 285

Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile

290 295 300

Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr

305 310 315 320

Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu

325 330 335

His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg

340 345 350

Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val

355 360 365

Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His Val

370 375 380

Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu Phe Cys

385 390 395 400

Val Leu

<210> 18

<211> 277

<212> PRT

<213>Homo sapiens

<400> 18

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1 5 10 15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

20 25 30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

35 40 45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

50 55 60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65 70 75 80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

85 90 95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

100 105 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

115 120 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

130 135 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145 150 155 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

165 170 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

180 185 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu

195 200 205

Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val

210 215 220

Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu

225 230 235 240

Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly

245 250 255

Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser

260 265 270

Thr Leu Ala Lys Ile

275

<210> 19

<211> 183

<212> PRT

<213>Homo sapiens

<400> 19

Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg

1 5 10 15

Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln

20 25 30

Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser

35 40 45

Ala Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val

50 55 60

Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln

65 70 75 80

Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn

85 90 95

Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu

100 105 110

Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln

115 120 125

Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr

130 135 140

Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu

145 150 155 160

Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn

165 170 175

Pro Gly Glu Phe Cys Val Leu

180

<210> 20

<211> 410

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 20

Leu Ala Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

65 70 75 80

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

100 105 110

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

130 135 140

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Pro Gly Lys Glu Leu Leu Gly Gly Gly Ser Ile Lys Gln

225 230 235 240

Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu

245 250 255

Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg Gly His Gly

260 265 270

Gly Gly Ser Asn Ser Gln Val Ser His Arg Tyr Pro Arg Phe Gln Ser

275 280 285

Ile Lys Val Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu

290 295 300

Thr Ser Gln Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val

305 310 315 320

Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe

325 330 335

Ser Gln Glu Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro

340 345 350

Leu Phe Gln Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala

355 360 365

Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn

370 375 380

Thr Ser Leu Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile

385 390 395 400

His Gln Asn Pro Gly Glu Phe Cys Val Leu

405 410

<210> 21

<211> 223

<212> PRT

<213>Homo sapiens

<400> 21

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 22

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Arg Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 24

<211> 121

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 24

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 451

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 25

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 26

<211> 214

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 27

<211> 412

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthesis polypeptide "

<400> 27

Ala Pro Leu Ala Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys Glu Leu Leu Gly Gly Gly Ser Ile

225 230 235 240

Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His

245 250 255

Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg Gly

260 265 270

His Gly Gly Gly Ser Asn Ser Gln Val Ser His Arg Tyr Pro Arg Phe

275 280 285

Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe

290 295 300

Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn

305 310 315 320

Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly

325 330 335

Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu

340 345 350

Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met

355 360 365

Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr

370 375 380

Asp Asn Thr Ser Leu Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile

385 390 395 400

Leu Ile His Gln Asn Pro Gly Glu Phe Cys Val Leu

405 410

<210> 28

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<221>Source

<223>/ annotation=" the description of artificial sequence：Synthetic peptide "

<400> 28

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims (28)

1. it is a kind of in subject treat solid tumor method, this method include to the subject give anti-PD-L1 antibody or its Antigen-binding fragment, anti-CTLA-4 antibody or its antigen-binding fragment and OX40 activators.

2. the method as described in claim 1, wherein the OX40 activators are in OX40 ligand fusion proteins or anti-OX40 antibody One or more.

3. method as claimed in claim 2, wherein the OX40 ligand fusion proteins are MEDI6383.

4. such as the method any one of claim 1-3, wherein the anti-PD-L1 antibody is MEDI4736.

5. such as the method any one of claim 1-4, the wherein anti-CTLA-4 antibody is Sibutramine Hydrochloride wood monoclonal antibody.

6. a kind of method that solid tumor is treated in subject, this method includes giving MEDI4736 or its antigen to the subject Binding fragment, Sibutramine Hydrochloride wood monoclonal antibody or its antigen-binding fragment and MEDI6383.

7. such as the method any one of claim 1-6, wherein this, which is given, increases survival period.

8. method as claimed in claim 7, wherein compared to single MEDI4736, single Sibutramine Hydrochloride wood monoclonal antibody or single MED6383's gives, and this gives the increase for causing survival period.

9. method as claimed in claim 7, wherein compared to MEDI4736 and Sibutramine Hydrochloride wood monoclonal antibody, MEDI4736 and MEDI6383, And Sibutramine Hydrochloride wood monoclonal antibody and MEDI6383 give, this gives the increase for causing survival period.

10. method as claimed in any one of claims 1-9 wherein, wherein this, which is given, reduces gross tumor volume.

11. method as claimed in claim 10, wherein compared to single MEDI4736, single Sibutramine Hydrochloride wood monoclonal antibody or independent MED6383 give, this gives the reduction for causing gross tumor volume.

12. method as claimed in claim 10, wherein compared to MEDI4736 and Sibutramine Hydrochloride wood monoclonal antibody, MEDI4736 and MEDI6383 and Sibutramine Hydrochloride wood monoclonal antibody and MEDI6383 are given, and this gives the reduction for causing gross tumor volume.

13. such as the method any one of claim 1-12, the wherein anti-PD-L1 antibody or its antigen-binding fragment Give is carried out by intravenous infusion.

14. such as the method any one of claim 1-13, the wherein anti-CTLA-4 antibody or its antigen-binding fragment Give is carried out by intravenous infusion.

15. such as the method any one of claim 1-14, wherein MEDI6383 or giving for its active fragment are to pass through What intravenous infusion was carried out.

16. such as the method any one of claim 1-15, the wherein solid tumor is oophoroma, breast cancer, colorectum Cancer, prostate cancer, cervical carcinoma, uterine cancer, carcinoma of testis, carcinoma of urinary bladder, head and neck cancer, melanoma, cancer of pancreas, clear-cell carcinoma or lung Cancer.

17. method as claimed in claim 16, the wherein solid tumor are triple negative breast cancers.

18. method as claimed in claim 17, the wherein solid tumor are non-small cell lung cancers.

19. method as claimed in claim 18, the wherein solid tumor are squamous or non-squamous non-small cell lung cancer.

20. such as the method any one of claim 1-19, the wherein subject is people patient.

21. a kind of pharmaceutical composition, anti-PD-L1 antibody or its antigen-binding fragment of the pharmaceutical composition comprising effective dose, Anti- CTLA-4 antibody or its antigen-binding fragment and OX40 activators and pharmaceutically acceptable excipient.

22. pharmaceutical composition as claimed in claim 21, wherein the OX40 activators are MEDI6383.

23. such as the pharmaceutical composition any one of claim 21-22, wherein the anti-PD-L1 antibody is MEDI4736.

24. such as the pharmaceutical composition any one of claim 21-23, the wherein anti-CTLA-4 antibody is that Sibutramine Hydrochloride wood is single It is anti-.

25. a kind of pharmaceutical composition, the pharmaceutical composition includes the MEDI4736 or its antigen-binding fragment, Sibutramine Hydrochloride wood of effective dose Monoclonal antibody or its antigen-binding fragment and MEDI6383 and pharmaceutically acceptable excipient.

26. such as the pharmaceutical composition any one of claim 21-25, the pharmaceutical composition is formulated for intravenously Give.

27. a kind of kit, the kit is comprising the pharmaceutical composition as any one of claim 21-26 and for controlling Treat the specification of cancer.

It is used for 28. kit as claimed in claim 27, the wherein kit further include as claimed in claim 1 The specification of the kit is used in method.